1

Natural variability of hepatic biomarkers in Mediterranean deep-sea organisms

Samuel Koenig a,b

* and Montserrat Solé a,

a Instituto de Ciencias del Mar (ICM-CSIC). Paseo Marítimo de la Barceloneta 37-49,

08003 Barcelona, Spain.

b Instituto de Diagnóstico Ambiental y Estudios del Agua (IDAEA-CSIC). Jordi Girona

18, 08034 Barcelona, Spain.

*Corresponding author. Tel.: +34 93 230 95 00; fax: +34 93 230 95 55. E-mail address:

koenig@icm.csic.es

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Abstract

Biomarker assays are widely used as proxies for contaminant-induced effects in aquatic

organisms. However, in many cases, their intrinsic natural variability due to exogenous

and endogenous factors makes the interpretation of biomarker data difficult. In the

present study, we investigated the natural fluctuations of six hepatic biomarkers, namely

ethoxyresorufin-O-deethylase (EROD) in fish and pentoxyresorufin-O-deethylase

(PROD) in crustacea, catalase (CAT), carboxylesterase (CbE), glutathione-S-transferase

(GST), total glutathione peroxidase (GPX) and glutathione reductase (GR) in two deep-

sea fish species, namely Alepocephalus rostratus and Lepidion lepidion and the decapod

crustacean Aristeus antennatus. The NW Mediterranean deep-sea environment is

characterized by very stable temperature and salinity conditions, allowing the exclusion

of these two factors as potential sources of interference with biomarker activities.

Biomarker results exhibited a clear influence of reproductive processes on enzyme

activities, in particular in A. rostratus, which presented a pronounced seasonal pattern

linked to variations in the gonadosomatic index (GSI). In addition, other factors such as

food availability may also have influenced the observed variability, in particular in

specimens of L. lepidion, which did not exhibit variations in reproductive activity

throughout the sampling period. Depth-related variability did not exhibit a clear trend

and fluctuations across sampling depths were not attributable to any specific factor.

Body size had also a significant influence on some biomarkers, although allometric

scaling of certain enzyme activities appears to be species-specific. The present work has

thus shown that despite the lack of fluctuations of abiotic parameters such as

temperature and salinity, biomarker activities in deep-sea organisms still exhibit

significant variability, mainly as a result of reproductive processes and food availability.

Keywords: biomarkers, deep-sea, natural variability, seasonality, xenobiotic

metabolism, antioxidant enzymes, NW Mediterranean

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1. Introduction

Biomarkers have been defined as measures of changes in biological parameters

resulting from contaminant exposure and their use has been advocated as a means to

provide early detection of exposure and adverse effects of pollutants on aquatic

organisms (Peakall, 1992). In this context, a number of parameters have been

investigated to assess chemical-induced disturbances of biological functions (van der

Oost et al., 2003). However, it is very unlikely that a single biomarker response can

unequivocally provide a measure of environmental degradation and the use of a suite of

biomarkers has thus been advocated (Handy et al., 2003; Galloway et al., 2004). In

particular, biomarkers are susceptible to natural variability due to abiotic (e.g.

temperature, salinity, dissolved oxygen) and biotic factors (e.g. gender, age, size,

reproductive stage) (Whyte et al., 2000; van der Oost et al., 2003; Martínez-Álvarez et

al., 2005). These confounding factors can sometimes mask the effect of contaminant-

induced stress signals and impede the interpretation of biomarker results (Sheehan and

Power, 1999). For the practical application of biomarkers there are several options to

minimize their variability such as the careful experimental design of field studies, data

normalization and the characterization of confounding environmental and biological

factors (Flammarion and Garric, 1999; Handy et al., 2003; Sanchez et al., 2008). To be

able to implement biomarkers in environmental monitoring studies it is thus crucial to

previously establish baseline levels and characterize the natural variability of these

assays.

The suite of hepatic biomarkers used in the present study included xenobiotic

metabolism enzymes such as ethoxyresorufin-O-deethylase (EROD) in fish and

pentoxyresorufin-O-deethylase (PROD) in crustacea, glutathione-S-transferases (GST)

and carboxylesterases (CbE) as well as enzymatic antioxidant defenses, such as catalase

(CAT), glutathione-peroxidase (GPX) and glutathione reductase (GR). The EROD and

PROD assays are commonly used as proxies for CYP1A- and CYP2B- mediated phase I

metabolism, respectively (Goksøyr and Förlin, 1992; Koenig et al., 2012b), which is

responsible for the biotransformation (mainly oxidation) of numerous endogenous and

exogenous compounds in fish and crustacea, respectively. CbEs are also categorized as

phase I drug metabolizing enzymes involved in the hydrolysis of ester-containing

chemicals (Satoh and Hosokawa, 2006; Wheelock et al., 2008). GST enzymes form part

of the phase II metabolism, which involves the conjugation of the xenobiotic compound

4

or its metabolite with an endogenous molecule (e.g. glutathione) to facilitate excretion

(Nimmo, 1987). Moreover, these enzymes can also function as antioxidant enzymes

catalyzing the reduction of organic hydroperoxides (Wang and Ballatori, 1998). Other

antioxidant enzymes that inhibit the formation of reactive oxygen species (ROS) are

CAT, which is responsible for the reduction of H2O2, GPX, which catalyzes the

reduction of peroxides to their corresponding alcohols and GR, which maintains the

homeostasis between GSH/GSSG under oxidative stress conditions (Winston and Di

Giulio, 1991).

The Mediterranean deep-sea (> 400m) is characterized by fairly stable temperatures,

and salinity (Danovaro et al., 2010), although episodic events can cause pronounced

fluctuations in hydrological parameters and particle fluxes (Heussner et al., 2006;

López-Fernández et al., 2012). In particular, episodic dense-shelf water cascading

(DSWC) events have been shown to take place in the NW Mediterranean every 6-10

years (Canals et al., 2006; Company et al., 2008). During these events, cold shelf water

masses cascade down the continental slope transporting large amounts of sediment and

organic matter, resulting in an increased particle-associated contaminant input to the

deep-sea environment (Salvadó et al., 2012). In addition, previous work has shown that

deep-sea organisms dwelling within submarine canyons in the NW Mediterranean are

particularly at risk of experiencing adverse contaminant-induced effects (Koenig et al.,

2012a). These findings further stress the need for the implementation of regular

environmental monitoring studies in these areas.

The species selected for the present study include the deep-sea fish Alepocephalus

rostratus (Alepocephaliform), Lepidion lepidion (Gadiform) and the crustacean Aristeus

antennatus (Decapoda). A. rostratus can be found in the eastern Atlantic and north-

western Mediterranean from 500 m up to 2300 m depth, with maximum aggregations at

midslope depths between 1000 m and 1450 m (Morales-Nin et al., 1996). L. lepidion is

mainly found in the NW Mediterranean and has a wide bathymetric distribution (500-

2300 m), although it is most abundant at the lower depths of the continental slope

(Rotllant et al., 2002). A. Antennatus is a eurybathic species with a known depth range

from 80 m to 3300 m, which can be found throughout the Mediterranean Sea and along

the NW African coast. This shrimp species is also one of the most valuable fishery

resources in the Mediterranean (Company et al., 2008). All three species have been

previously used in environmental monitoring studies conducted in Mediterranean deep-

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sea habitats (Escartin and Porte, 1999; Porte et al., 2000; Antó et al., 2009; Solé et al.,

2009; 2010).

The main objective of the present study was to characterize baseline levels and the

natural variability of selected hepatic biomarkers in two deep-sea fish and a crustacean

species. We determined EROD or PROD, respectively, GST, CbE, CAT, GPX and GR

activities in the fish A. rostratus and L. Lepidion and the crustacean A. antennatus from

four seasonal sampling periods and different sampling depths. Furthermore, the

relationship between biomarker activities and biological parameters (e.g. size, gender,

sexual maturity) of the sampled organisms was investigated.

2. Material and Methods

2.1. Collection of animals and sampling site

Seasonal sampling cruises were carried out off the coast of Blanes, north-western

Mediterranean (41º15’N 2º50’E) onboard the R/V Garcia del Cid in winter (February),

spring (May), summer (September) and autumn (November) in 2009. Fish were caught

using a OTMS otter trawl (Sardà et al., 1998) at various water depths ranging from 900

m to 2000 m (Figure 1). The OTMS is a benthic trawling net with a cod-end mesh size

of 40 mm fitted with two divergent doors and a single warp cable. Total trawl times,

including net deployment and retrieval, ranged between 1.5-3 h depending on sampling

depth (winch speed 70 m/s), with bottom haul times of 40-60 min. Only animals

dissected within 2 h of net retrieval were considered for biochemical analyses. Body

size, weight and sex were recorded and the liver/hepatopancreas was dissected and

frozen in liquid nitrogen and stored at -80 °C until further analysis. GSI values were

only available for A. rostratus as the gonad weight could not be recorded for L. lepidion

and A. antennatus due to technical limitations onboard the vessel. Number of

individuals sampled for each season and depth are shown in Table 1 and Table 3,

respectively.

2.2. Sample preparation

A portion of liver/hepatopancreas (approx 0.5 g) was homogenized 1:4 (w:v) in a 100

mM phosphate buffer pH 7.4 containing for fish liver 150 mM KCl, 1 mM dithiothreitol

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(DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMFS), 1 mM

ethylenediaminetetraacetic acid (EDTA) and for crustacean hepatopancreas 100mM

KCl, 1mM EDTA, 0.1 mM phenanthroline and 0.1 mg/L trypsin inhibitor. The

homogenate was centrifuged at 10,000 g for 30 min and the obtained supernatant (S9)

was stored at -80 °C until further biochemical analyses.

2.3. Biochemical analysis

All assays were carried out in triplicate at 25 °C in 96-well format using a TECAN™

Infinite M200 microplate reader. For each assay, blank samples were analyzed in

triplicate, which were used to correct for background activity. Prior to analysis, assay

conditions were optimized for each species by determining the appropriate dilution of

the S9 supernatant (protein content 5-10 mg/mL) for each assay to ensure constant

linearity of the measured activity (dilutions for each species shown below in parenthesis

for each assay). All reaction mixtures, except for catalase, contained 100 mM phosphate

buffer pH 7.4.

Catalase (CAT) activity was measured in a UV-transparent microplate (Greiner UV-

Star®) as absorbance decrease at 240 nm for 1 min using 50 mM H2O2 as substrate (ε =

40 mol-1

* cm-1

) and a 100 mM phosphate buffer pH 6.5 (Aebi, 1974 ). Sample volume

used was 10 µL (Ar 1:400, Ll 1:200, Aa 1:2) in a total volume of 210 µL.

Glutathione reductase (GR) activity was measured as decrease in absorbance at 340 nm

for 3 min using 0.09 mM nicotinamide adenine dinucleotide phosphate (NADPH) (ε =

6.22 * mmol-1

* cm-1

) and 0.9 mM oxidized glutathione (GSSG) as substrate (Carlberg

and Mannervik, 1985). Sample volume used was 20 µL (not diluted) in a total volume

of 200 µL.

Total glutathione-peroxidase (GPX) activity was determined as decrease in absorbance

at 340 nm during 3 min using 2.5 mM reduced glutathione (GSH), 1 mM glutathione

reductase (GR), 0.625mM cumene hydroperoxide (CHP) and 0.3mM NADPH (ε = 6.22

* mmol-1

* cm-1

) (Günzler and Flohe, 1985). Sample volume used was 10 µL (not

diluted) in a total volume of 240 µL.

Glutathione-S-transferase (GST) activity was measured as increase in absorbance at

340 nm for 3 min using 1 mM 1-chloro-2,4- dinitrobenzene (CDNB) (ε = 9.6 * mmol-1

*

7

cm-1

) and 1 mM GSH as substrate (Habig et al., 1974). Sample volume used was 25 µL

(Ar 1:20, Ll 1:20, Aa 1:20) in a total volume of 225 µL.

Carboxylesterase (CbE) activity was determined as increase in absorbance at 405 nm

during 5 min using 0.18 mM 5,5-dithio-bis-2-nitrobenzoate (DTNB) (ε = 13.6 * mmol-1

* cm-1

) and 0.67 mM S-phenylthioacetate as substrate (Ellman et al., 1961). Sample

volume used was 25 µL (Ar 1:5, Ll 1:5, Aa 1:20) in a total volume of 225 µL.

7-Ethoxyresorufin-O-deethylase (EROD) activity for fish and 7-Pentoxyresorufin-O-

deethylase (PROD) activity in crustacea were measured kinetically as increase in

fluorescence at 537 nm excitation and 583 nm emission over 10 min based on the

procedure by Burke and Mayer (1974). Substrates used include 7-ethoxyresorufin (3

µM) and 7-pentoxyresorufin (5 µM), respectively and 0.2 mM NADPH with a seven-

point curve of resorufin sodium salt standard. Sample volume used was 50 µL (not

diluted) in a total volume of 250 µL.

Protein content was determined according the method by (Bradford, 1976), using

bovine serum albumin as standard (BSA 0.1–1 mg/ml). Sample volume used was 10 µL

(Ar 1:20, Ll 1:40, Aa 1:20) in a total volume of 260 µL.

2.4. Statistical analysis

Data were checked for normality (Shapiro-Wilk’s test) and homogeneous variance

(Levene’s test) and were log10(x)-transformed for parametric t-test/ANOVA/ANCOVA

analyses followed by Tukey´s HSD test for multiple comparisons. Correlations were

determined using Pearson’s correlation coefficient. Differences at the 5% significance

level were considered significant. In the case of significant correlations between enzyme

activities and body size, ANCOVA tests were performed introducing size as covariable

in the model. The interaction factors (e.g. Sex*Size) were only included in the model if

significant (unequal slopes) (Engqvist, 2005).

3. Results

Temperature and salinity exhibited very little seasonal fluctuations with a maximum

variation of 0.2 ºC temperature and 0.04 PSU salinity across seasons. The depth profile

exhibited a slight increase in salinity from 900 m to 1500 m depth (approx. 0.3 PSU),

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while temperature was 0.2 ºC higher at 900 m compared to all lower depths (Tecchio et

al., 2012).

3.1. Biomarkers in Alepocephalus rostratus

Male and female A. rostratus differed significantly in size, with females being larger

than males (t-test, t = 4.79, P < .0001). Moreover, males and females exhibited

significant seasonal differences in body size and GSI (Table 1). Because some

biomarker activities varied between sexes (i.e. EROD and GST), seasonal comparisons

were conducted for males and females separately. Due to the segregated sex distribution

of A. rostratus at different depths, comparisons among depths could not be performed

for this species. All statistical results are given in Table 2.

Overall, a negative correlation was observed between EROD, CbE and GPX activity

and body size. EROD, GST, CbE and GPX differed significantly between sexes,

although in the case of CbE and GPX this difference was mainly due to size. Moreover,

EROD and GST activities were significantly correlated negatively with the GSI in

females, while CbE and CAT exhibited a negative correlation with the GSI in both

sexes. All biomarkers, except CAT, exhibited seasonal variations in females, whereas

CbE, GR and CAT fluctuated significantly in males (Figure 2 and Table 2).

3.2. Biomarkers in Lepidion lepidion

There was no significant difference in size between male and female L. lepidion, but

size differed among seasons (Table 1) and depths (Table 3). Seasonal variation was

investigated in samples from 1200 m depth and the depth-related variability was

assessed in fish collected during autumn from 900 m to 2000 m depth (Table 3). Details

for statistical analyses are also given in Table 2.

Out of the six biomarkers analyzed, only GST activity exhibited a significant

relationship with body size. Moreover, no differences in enzyme activities were

detected between sexes and results are thus presented together regardless of sex.

However, all enzyme activities, except GPX, varied seasonally (Figure 3). EROD and

CbE activity exhibited a peak in spring, while GST, CAT and GR activities were lower

during summer (Figure 3). Moreover, CbE and GR activities differed significantly

among sampling depths, while the depth-related variations in GST activity were due to

differences in body size (Table 3).

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3.3. Biomarkers in Aristeus antennatus

Carapace size differed significantly between sexes and female A. antennatus were

significantly larger than males (t-test, t = 8.47, P < .0001). Moreover, carapace size also

differed among seasons (Table 1) and depths (Table 3). Seasonal variations were

assessed combining data from several depths (i.e. 900 m, 1200 m and 1500 m), while

the effect of depth was determined in samples collected in autumn (Table 3). Details for

statistical analyses are also given in Table 2.

CbE and GR activities exhibited a negative correlation with carapace size, whereas for

CAT it was positive. These three biomarkers presented differences in activities between

sexes, but in all three cases the variability was attributed to differences in size.

PROD, CbE, GPX and CAT presented a peak in activity in spring, while a significant

decline in GST activity was observed in autumn (Figure 4). ANCOVA results indicated

that seasonal variations in CbE activity were mainly due to differences in body size.

PROD, GST, CbE and GPX varied significantly among sampling depths, although no

clear pattern was observed (Table 3).

4. Discussion

Seasonal and depth-related fluctuations of water temperature and salinity in this deep-

sea environment were minimal and up to two orders of magnitude lower than the

variations of these parameters reported for studies that observed seasonal variations of

biomarkers in fish from coastal areas of the Baltic Sea (Kopecka and Pempkowiak,

2008), the Adriatic Sea (Pavlović et al., 2010) or the Arctic Ocean (Nahrgang et al.,

2010). Hence, the influence of these two abiotic parameters on the variability of

biomarker activities among seasons and sampling depths is likely to be negligible.

Furthermore, as the biomarkers included in the present study are used as proxies for

contaminant exposure, variations in contamination levels could also have influenced the

presented results. However, as shown by Gomez-Gutiérrez et al. (2007), organic

contaminant levels in Mediterranean offshore sediments (> 1000 m) are considered

background contamination levels for the region due to the remoteness from pollution

sources and exhibit low temporal variability. Moreover, although the transfer of

pollutants from NW Mediterranean surface waters to the deep-sea has been shown to

10

increase during episodic dense-shelf water cascading (DSWC) events (Salvadó et al.,

2012), no such event occurred in 2009 when the present study was conducted, with the

last one registered during the winter 2005/06. In addition, chemical analyses of biota

from the study area did not reveal any seasonal changes in contamination levels

(author’s unpublished data) and it is thus assumed that biomarker results from the

present work are likely not affected by variations in pollution levels.

4.1. Alepocephalus rostratus

Numerous studies have shown that differences in sex and body size can influence

enzymatic activities and complicate the interpretation of biomarker results (van der Oost

et al., 2003). As female A. rostratus were significantly larger than males, it is important

to determine whether the observed gender-related differences were actually due to

differential enzyme activities or resulted from the above-mentioned sexual dimorphism.

In the present study, EROD, GST, CbE and GPX activities varied significantly between

sexes and all, except GST, were higher in males than females. Moreover, significant

overall correlations with body size were observed for all the above-mentioned

biomarkers, except GST, suggesting that for the latter enzyme, differences between

sexes cannot be attributed to size. The gender-dependent EROD activity, with

significantly higher activities in liver of male fish, is a well documented phenomenon

(Whyte et al., 2000). CYP1A-related EROD activity has been shown to be suppressed

in mature females by 17β-estradiol and a decline of CYP1A activity is usually observed

from the onset of ovulation until spawning (Whyte et al., 2000). Moreover, EROD

activity showed a negative relationship with GSI in females, which is consistent with

other studies (Flammarion et al., 1998; Kopecka and Pempkowiak, 2008). Although

mature individuals of A. rostratus were found all year round, a peak in maturation

usually occurs from summer to autumn, when spawning activity is highest (Morales-

Nin et al., 1996; Follesa et al., 2007). Accordingly, the individuals analyzed in the

present study presented the highest GSI during summer and autumn, while at the end of

the spawning period in spring the GSI was at its lowest (Table 1). In fact, the end of the

spawning period coincided with in an increase in EROD activity in females, reaching

activity levels similar to males. Moreover, the increase in GSI in summer coincided

with a decline in EROD activity in females, while seasonal variations of EROD were

absent in males.

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The conjugating enzyme GST also exhibited seasonal fluctuations only in females, a

trend that has also been reported in other studies (Ronisz et al., 1999). Furthermore,

females exhibited a negative correlation between GST activity and the GSI, suggesting

that female sex hormones might also influence this enzyme activity. In contrast to GST,

ANCOVA results indicated that gender-related differences in CbE activity were likely

due to the sexual dimorphism as, once adjusted for body size, CbE activity did not differ

between sexes. This size-dependence of CbE activity is in accordance with results

presented for other fish species such as the Senegalese sole, further supporting the

assumption that CbEs behave like other esterase enzymes (e.g. cholinesterases) and that

the activity decreases with increasing body size (Solé et al., 2012). Contrarily, CbE

activity in rainbow trout has been shown to be independent of body size (Barron et al.,

1999), suggesting that the allometric scaling of CbE activity is species-dependent.

Alpuche-Gual and Gold-Bouchot (2008) also reported a significant correlation with size

as well as gender-dependent differences in CbE activity in the reef fish Haemulon

plumieri, but the influence of size on the gender-related differences in CbE activity was

not addressed. CbE enzymes are involved in reproductive processes such as lipid

metabolism and bioinactivation of specific hormones (Leinweber, 1987), which

potentially explains the negative correlation between CbE and the GSI in both sexes and

the significant decline of CbE activity during summer, when the GSI was highest.

The difference in GPX activity between sexes was mainly due to the difference in body

size. However, the fact that seasonal variation of GPX activity was only observed in

females suggests that some sex-related factor potentially affected GPX activity, which is

consistent with previous studies (Ronisz et al., 1999; Sanchez et al., 2008). The

significant relationship of CAT activity with the GSI in both genders and the concordant

activity decline in summer are also in accordance with the observations made by Ronisz

et al.(1999) and suggest the influence of reproductive processes on CAT activity. In

contrast, GR activity was lowest in spring when reproductive activity is low and highest

in summer and autumn during the spawning period. Furthermore, it should be noted that

all glutathione-dependent enzymes, namely the antioxidant enzymes GPX and GR, as

well as GST, presented highest activities during autumn.

In addition to reproductive processes, food availability has also been shown to influence

biotransformation enzyme activities such as EROD and CbE (Leinweber, 1987; Bucheli

and Fent, 1995; Whyte et al., 2000) and antioxidant enzymes including GST, GPX, GR

12

and CAT (Martínez-Álvarez et al., 2005). In this context, the simultaneous study by

López-Fernández et al. (2012) on particle fluxes in the study area revealed a peak in

particulate matter input from autumn to spring, while during summer particle fluxes

were lower. However, although the influence of food availability on antioxidant

enzymes is well described, the direction of change of these responses (increase or

decrease) can be variable (Martínez-Álvarez et al., 2005). For instance, brown trout

(Salmo trutta) antioxidant defenses such as CAT, GPX and GR increased as a result of

food deprivation, while GST decreased (Bayir et al., 2011). Moreover, Pascual et al.

(2003) showed that the direction of variation of some antioxidant activities such as GPX

and GST may vary according to the level and duration of the food deprivation period.

The same study showed that an increased level of lipid peroxidation due to prolonged

starvation could have opposite effects on CAT and GR activities. This trend is also

apparent in the present study in which GR was significantly lower in spring than in

summer and CAT the other way round.

4.2. Lepidion lepidion

In contrast to A. rostratus, L. lepidion did not exhibit any sex-related differences in

biomarkers, which is consistent with the lack of sexual dimorphism in this species (i.e.

equal body size) and the fact that no fully mature individuals were caught throughout

the sampling period (all individuals were classified as maturity stage II). However, all

biomarker activities varied significantly among seasons, with most enzymes exhibiting

a decline in activity during summer, a peak in metabolizing enzymes EROD and CbE in

spring and high antioxidant activities in autumn. Hence, the seasonal variability of

enzymatic activities observed for L. lepidion is probably not related to fluctuations in

reproductive activity, but results from other factors such as the above-mentioned

variations in food availability. Indeed, higher particle fluxes in spring may be

responsible for the increase in biotransformation enzyme activities (i.e. EROD and

CbE) (Leinweber, 1987; Bucheli and Fent, 1995; Whyte et al., 2000), while the lower

antioxidant activities in summer could be related to lower particle fluxes during that

time. Coinciding with A. rostratus, antioxidant activities were elevated in autumn,

indicating that similar factors might affect these parameters in both species. Biomarkers

in specimens collected at different sampling depths only differed significantly in CbE

and GR activities and no clear trend was apparent.

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4.3. Aristeus antennatus

Biomarker data for A. antennatus exhibited gender-dependent activity for GPX, CbE

and CAT, although the inclusion of size in the model canceled out the effect of sex for

CbE and CAT. Hence, it seems that differences in CbE and CAT activities between

male and female shrimp result from the pronounced sexual dimorphism in this species.

These results are in accordance with previous findings that reported sex-related

differences in GPX, but not CAT activity in freshwater gammarids (Sroda and Cossu-

Leguille, 2011). In the present study, GPX and CAT exhibited opposite correlation

patterns with size, which is similar to observations in brain tissue of A. antennatus,

(Mourente and Díaz-Salvago, 1999). Furthermore, GPX and GR activities exhibited a

significant overall negative relationship with size, which is consistent with the general

idea of metabolic scaling of antioxidant activities as a result of decreasing oxygen

consumption (and associated ROS production) with increasing size (Amérand et al.,

2010).

A clear peak in GPX and CAT activities was observed during spring, coinciding with

the reproductive period of A. antennatus. Sexual development of adult A. antennatus

reaches its maximum from May to September, accompanied by increased molting

activity during this period (Demestre, 1995). Thus, the peak in antioxidant defenses

during spring (late May) might result from increased reproductive and associated

molting activity, which is in accordance with previous studies on other crustacean

species (Sroda and Cossu-Leguille, 2011). Moreover, a peak in CAT activity during

June 2007 was recorded in a previous study conducted on the same species (Antó et al.,

2009), supporting the idea of enhanced antioxidant activities during the reproductive

period. The increase in CAT activity was also more pronounced than for GPX activity.

This difference in response amplitude can potentially be explained by the fact that both

enzymes have complementary roles in hydrogen peroxide detoxification as well as

different cellular localizations (Janssens et al., 2000; Barata et al., 2005). A peak in

activity during spring was also evident for CbE, suggesting the involvement of CbE in

the sexual development of A. antennatus. In fact, CbEs are thought to be involved in the

regulation of physiological processes in crustaceans such as molting and reproduction

(i.e. catabolism of juvenile hormone) (Ezhilarasi and Subramoniam, 1984; Reddy et al.,

2004; Lee et al., 2011). Similarly, CYP450 enzymes are thought to be involved in

crustacean molting and reproductive processes (James and Boyle, 1998; Rewitz et al.,

14

2006) and PROD activity was also highest in spring, although not statistically

significant due to high inter-individual variability. Moreover, the lack of GST activity

increase during spring is consistent with previous results reporting that molting did not

alter GST activity in crabs (Hotard and Zou, 2008),.

Contrasts among sampling depths exhibited significantly lower PROD, CbE and GPX

activities at 2000 m compared to 1200 m and 1500 m depth. As mentioned previously,

these three enzymes are likely influenced by the reproductive and molting cycle.

Moreover, reproductively active adult A. antennatus have been shown to aggregate at

shallower depths (Sardà et al., 2004). Hence, it is possible that individuals caught at

shallower depths exhibit higher reproductive and associated molting activities than

deeper-dwelling specimens. In contrast, GST activity was higher at greater depths,

confirming the lack of influence of molting on GST.

5. Conclusions

The present work has shown that despite the lack of seasonal and depth-related

fluctuations in temperature and salinity, which are characteristic for most deep-sea

habitats, biomarker activities in deep-sea organisms still exhibit significant variability.

All three species experienced seasonal variations of enzyme activities as a result of

fluctuations of endogenous factors such as reproductive processes and/or exogenous

factors such as food availability. However, the fact that A. rostratus exhibited higher

gender-related seasonal variability than L. lepidion indicates that L. lepidion might be a

more adequate sentinel species for future monitoring studies. Furthermore, allometric

scaling of enzymatic activities was not consistent among species, indicating that these

relationships need to be investigated on a species-specific level. In this context, the use

of appropriate statistical analyses such as the ANCOVA test, which allow the

assessment of the covariation of biomarkers with body size, is highly recommended.

Acknowledgements

The present study was funded by the Spanish Science and Technology Ministry projects

PROMETEO (CTM2007-66316-C02-02/MAR), BIOFUN (CTM2007-28739-E/MAR)

15

and the HERMIONE project (EC-FP7 contract number 226354). Samuel Koenig holds

a PhD grant (AFR 08/067) from the Fonds National de la Recherche (FNR),

Luxembourg. The authors wish to thank Sofia Vega and Elisa Salas for their help with

biochemical analyses, as well as the DeepMed Research Group (ICM-CSIC) and the

R/V Garcia del Cid (CSIC) crew for helping with sampling. The map of the study area

was done by J.A. Garcia (ICM-CSIC).

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Table 1 Seasonal variation of biological parameters of selected deep-sea species. Values are shown as

mean ± S.E.M. Different letters denote significant differences between seasons based on Tukey’s

HSD multiple comparisons (P < 0.05).

Species Parameter Sex Winter Spring Summer Autumn

A. rostratus Sample size M 8 8 7 9

F 7 9 9 9

Size (mm) M 298.4 ± 16.2ab

326.4 ± 13.0a

301.3 ± 9.1ab

265.8 ± 13.6b

F 338.5 ± 15.7b

387.1 ± 4.1a

338.8 ± 5.1b

315.9 ± 14.7b

GSI (%) M 1.09 ± 0.18c

1.45 ± 0.33c

7.56 ± 0.85a

4.91 ± 0.70b

F 2.08 ± 0.75b 1.02 ± 0.15

b 7.57 ± 1.81

a 2.85 ± 1.01

b

L. lepidion Sample size 8 10 10 10

Size (mm) 161.1 ± 6.2b

207.0 ± 4.2a

201.8 ± 3.5a

197.3 ± 5.3a

A. antennatus Sample size 30 30 30 30

Size (mm) 45.1 ± 2.3a

47.9 ± 2.0a

41.3 ± 2.2a

32.2 ± 1.9b

Table 2 Details for statistical analyses of biomarker data. Table shows Pearson’s correlation for size and GSI, as well t-test/ANCOVA for contrasts between sex, and

ANOVA/ANCOVA for seasonal and depth comparisons. In the case of significant correlations between enzyme activities and size, ANCOVA tests were performed introducing size as

covariable in the model. Effect size is reported as partial eta-squared values (η2) based on Type III sum of squares.

Species Factor EROD GST CbE GPX GR CAT

Alepocephalus rostratus

Size (n = 120) R = -0.37*** n.s. R = -0.26** R = -0.38*** n.s. n.s.

Sex (n = 120) ANCOVA*** F = 20.19 t-test* t = 2.11 ANCOVA** F = 5.23 ANCOVA*** F = 12.14 t-test n.s. t-test n.s.

Sex*** η2 = 0.11 Sex * η2 = 0.03 Sex n.s. Sex* η2 = 0.03 Sex n.s. Sex n.s.

Size* η2 = 0.04 Size n.i. Size* η2 = 0.04 Size** η2 = 0.08 Size n.i. Size n.i.

GSI Males (n = 60) n.s. n.s. R = -0.31* n.s. n.s. R = -0.62***

Females (n = 60) R = -0.37* R = -0.48** R = -0.30* n.s. n.s. R = -0.55***

Season Males ANCOVA n.s. ANOVA n.s. ANCOVA*** F = 9.43 ANCOVA n.s. ANOVA* F = 3.49 ANOVA*** F = 8.84

1200 m Season n.s. Season n.s. Season*** η2 = 0.54 Season n.s. Season* η2 = 0.27 Season*** η2 = 0.49

(n = 32) Size n.s. Size n.s. Size n.s.

Females ANCOVA* F = 3.48 ANOVA*** 8.90 ANCOVA* F = 5.75 ANCOVA*** F = 7.18 ANOVA** F = 5.10 ANOVA n.s.

1500 m Season* η2 = 0.48 Season*** η2 = 0.48 Season* η2 = 0.28 Season** η2 = 0.34 Season** η2 = 0.35 Season n.s.

(n = 34) Size n.s. Size n.s. Size n.s.

Lepidion lepidion EROD GST CbE GPX GR CAT

Size (n = 78) n.s. R = 0.39*** n.s. n.s. n.s. n.s.

Sex (n = 78) t-test n.s ANCOVA n.s t-test n.s t-test n.s t-test n.s t-test n.s Season 1200 m ANOVA*** F = 14.18 ANCOVA*** F = 9.57 ANOVA*** F = 47.58 ANOVA*** F = 10.29 ANOVA*** F = 14.03 ANOVA*** F = 10.08

(n = 38) Season*** η2 = 0.56 Season*** η2 = 0.49 Season*** η2 = 0.81 Season*** η2 = 0.49 Season*** η2 = 0.55 Season*** η2 = 0.47

Size n.s.

Depth autumn ANOVA n.s. ANCOVA* F = 3.01 ANOVA*** F = 24.87 ANOVA n.s. ANOVA** F = 4.11 ANOVA n.s.

(n = 50) Depth n.s. Depth n.s. Depth*** η2 = 0.69 Depth n.s. Depth** η2 = 0.27 Depth n.s.

Size** η2 = 0.17

Aristeus antennatus PROD GST CbE GPX GR CAT

Size (n = 138) n.s. n.s. R = -0.40*** n.s. R = -0.23** R = 0.29**

Sex (n = 138) t-test n.s. t-test n.s. ANCOVA*** F = 13.26 t-test n.s. ANCOVA* F = 3.80 ANCOVA** F = 5.57

Sex n.s. Sex n.s. Sex n.s.

Size*** η2 = 0.13 Size* η2 = 0.04 Size* η2 = 0.05

Season (n = 120) ANOVA n.s. ANOVA*** F = 10.36 ANCOVA*** F = 21.82 ANOVA*** F = 6.34 ANCOVA n.s. ANCOVA*** F = 17.79

Season n.s. Season*** η2 = 0.21 Season*** η2 = 0.22 Season*** η2 = 0.14 Season n.s. Season*** η2 = 0.59

Size*** η2 = 0.35 Size n.s. Size n.s.

Depth autumn ANOVA* F = 3.24 ANOVA*** F = 6.30 ANCOVA*** F = 18.12 ANOVA*** F = 12.26 ANCOVA n.s. ANCOVA n.s.

(n = 48) Depth* η2 = 0.24 Depth*** η2 = 0.37 Depth*** η2 = 0.32 Depth*** η2 = 0.54 Depth n.s. Depth n.s.

Size*** η2 = 0.09 Size n.s. Size n.s.

* P < 0.05, ** P < 0.01, *** P < 0.001

n.s.; not significant (P > 0.05)

Table 3 Biomarker data (mean ± S.E.M.) for the fish Lepidion lepidion and the crustacean Aristeus antennatus sampled at different depths during autumn 2009. All

activities are expressed as nmol·min-1

·mg protein-1

except for EROD (pmol·min-1

·mg protein-1

), PROD (fmol·min-1

·mg protein-1

) and CAT (mmol·min-1

·mg protein-1

).

Species Depth (m) Size (mm) EROD GST CbE GPX GR CAT

L. lepidion

n = 10 900 185.9 ± 5.8 b 8.2 ± 2.7 335.1 ± 22.5 50.1 ± 2.2 a 51.9 ± 2.9 3.6 ± 0.7 b 0.9 ± 0.1

n = 10 1200 197.3 ± 5.3 b 4.0 ± 0.7 301.6 ± 18.4 24.2 ± 1.4 b 45.5 ± 2.2 6.3 ± 0.5 a 1.2 ± 0.2

n = 10 1500 217.6 ± 14.4 ab 4.1 ± 2.2 345.2 ± 14.9 31.8 ± 3.2 b 34.6 ± 1.7 3.8 ± 0.4 b 1.0 ± 0.1

n = 10 1700 244.8 ± 16.9 a 4.1 ± 0.9 469.2 ± 40.2 35.4 ± 3.6 b 43.1 ± 3.4 4.0 ± 0.3 ab 0.9 ± 0.1

n = 10 2000 224.0 ± 10.3 ab 3.9 ± 1.1 387.1 ± 43.2 75.3 ± 7.9 a 48.5 ± 2.6 4.3 ± 0.5 ab 1.1 ± 0.1

Depth (m) Size (mm) PROD GST CbE GPX GR CAT

A. antennatus

n = 10 900 43.0 ± 3.1 a 155.7 ± 16.9 ab 67.5 ± 11.4 b 360.8 ± 23.6 a 138.6 ± 8.0 c 0.9 ± 0.3 7.7 ± 1.2

n = 10 1200 29.0 ± 1.4 b 204.0 ± 20.6 a 89.2 ± 21.5 b 700.1 ± 69.9 a 220.2 ± 20.2 a 1.3 ± 0.2 n.a.

n = 10 1500 24.5 ± 1.6 b 197.5 ± 62.5 a 241.4 ± 70. a 795.4 ± 81.0 a 310.8 ± 48.5 a 1.8 ± 0.4 3.1 ± 1.4

n = 8 1700 26.6 ± 2.4 b 183.5 ± 79.7 ab 268.6 ± 88.8 a 464.1 ± 34.8 ab 208.8 ± 19.2 ab 1.6 ± 0.4 6.9 ± 0.7

n = 10 2000 33.5 ± 3.1 ab 101.7 ± 15.4 b 118.4 ± 20.3 ab 306.9 ± 39.9 b 149.2 ± 13.3 bc 1.5 ± 0.5 6.4 ± 1.4

Fig.1 Location of sampling sites off the coast of Blanes, NW Mediterranean. Map

created by J.A. García, using ESRI®

ArcMap™ 9.3 and bathymetric data from Canals et

al. (2004).

Fig.2 Seasonal variation of six hepatic biomarkers (mean ± S.E.M.) in male (black) and

female (grey) A. rostratus in winter (M: n = 8; F: n = 7), spring (M: n = 8; F: n = 9),

summer (M: n = 7; F: n = 9) and autumn (M: n = 9; F: n = 9). All activities are

expressed as nmol·min-1

·mg protein-1

except for EROD (fmol·min-1

·mg protein-1

).

Asterisks indicate biomarkers for which an ANCOVA test was performed due to a

significant correlation with size. For bars denoted by different letters biomarker values

differed significantly among seasons.

Fig.3 Seasonal variation of six hepatic biomarkers (mean ± S.E.M.) in Lepidion

lepidion rostratus in winter (n = 8), spring (n = 10), summer (n = 10) and autumn (n =

10). All activities are expressed as nmol·min-1·mg protein

-1 except for EROD

(pmol·min-1

·mg protein-1

) and CAT (µmol·min-1

·mg protein-1

). Asterisks indicate

biomarkers for which an ANCOVA test was performed due to a significant correlation

with size. For bars denoted by different letters biomarker values differed significantly

among seasons.

Fig.4 Seasonal variation of six hepatic biomarkers (mean ± S.E.M.) in A. antennatus in

winter (n = 30), spring (n = 30), summer (n = 30) and autumn (n = 30). All activities are

expressed as nmol·min-1

·mg protein-1

except for PROD (fmol·min-1

·mg protein-1

) and

CAT (mmol·min-1

·mg protein-1

). Asterisks indicate biomarkers for which an ANCOVA

test was performed due to a significant correlation with size. For bars denoted by

different letters biomarker values differed significantly among seasons.

Fig. 1

Fig. 2

Fig. 3

Fig. 4